Fig. 3: Cell-penetrating peptide-mediated delivery of ABE in vitro and in vivo.
a, Schematic cartoon of fluorescent rd12 reporter. Under the control of the CMV promoter, a gene expresses both mCherry and eGFP, with an intervening sequence from the Rpe65 rd12 genomic sequence. The rd12 mutation (c.130 C > T; p.R44X) prevents expression of eGFP, but successful base editing restores the reading frame and co-expression of mCherry and eGFP. b, Quantification of successful base editing of rd12 reporter cells, 48 h after co-transfection of CMV-ABE8e-NG and sgRNA plasmids. Three biological replicates with 2 analytical replicates each, mean ± s.d. c, Assessment by fluorescence microscopy (left) and flow cytometry (right) of ABE delivery to rd12 reporter cells, 48 h post treatment; mCherry and eGFP co-expression indicate successful delivery. Scale bar, 100 μm. d, Concentration dependence of efficiency of delivery of ABE in the presence of 2% (w/v) sucrose, 10% (w/v) sucrose, and of 0.5 µM ABE with 10% (w/v) sucrose and 6×His-CM18-PTD4 peptide. 1 µM corresponds to 224 µg ml−1. Two biological replicates with 2 analytical replicates each, mean ± s.d. e, ERG response curves from WT mice. f, ERG response curves from rd12 mice, 2 weeks post treatment with ABE RNP in the presence of high and low NaCl and sucrose, with and without 6×His-CM18-PTD4 peptide. The left curves represent the highest-responding eye from each treatment group, while the right curves represent a low-responding eye from each treatment group. g, Quantification of ERG responses (b-wave amplitude) from rd12 mice whose response curves are shown in e. ABE RNP concentrations are given in µM. 20 µM corresponds to 4.5 µg ABE RNP per eye, 36 µM to 8.1 µg per eye. At least 6 eyes, mean ± s.d., Kruskal–Wallis test with Dunn’s multiple comparisons test; *P < 0.05, **P < 0.01. $ # indicate data points corresponding to ERG response curves presented in panel f. h,i, Quantification of genomic DNA editing (h) and cDNA transcripts containing the edit (i) in ABE-treated mice whose responses are summarized in f. Colours are as in g. j, ERG response curves from rd12 mice, 2 weeks post treatment with ABE RNP with or without fused cell-penetrating peptides. k, Quantification of ERG responses (b-wave amplitude) from rd12 mice whose response curves are shown in j. $ and # indicate data points corresponding to ERG response curves presented in panel j.The mice received 4.5 µg ABE RNP per eye. At least 6 eyes, mean ± s.d.; Kruskal–Wallis test with Dunn’s multiple comparisons test; ***P < 0.001. l,m, Quantification of genomic DNA editing (l) and resulting proportion of transcripts containing the edit (m) in CPP-ABE-treated mice whose responses are summarized in j. The colours are as in k.
